The present invention relates to novel pyridine derivatives, processes for preparing them and pharmaceutical preparations containing them. The pyridine derivatives of the present invention inhibit IxcexaB kinase xcex2 (IKK-xcex2 or IKK-beta) activity, thus inhibit nuclear factor kappa B (NF-xcexaB) activity, and can be used for the prophylaxis and treatment of diseases associated with NF-xcexaB activity, in particular for the treatment of inflammatory diseases.
Nuclear factor kappa B (NF-xcexaB) belongs to a family of closely related homo- and hetero-dimeric transcription factor complexes composed of various combinations of the Rel/NF-xcexaB family of polypeptides. NF-xcexaB and related family members are involved in the regulation of more than 50 genes relating to immune and inflammatory responses ((Barnes P J, Karin M (1997) N Engl J Med 336, 1066-1071) and (Baeuerle P A, Baichwal V R (1997) Adv Immunol 65, 111-137)). In most cell types, NF-xcexaB is present as a heterodimer comprising a 50 kDa and a 65 kDa subunit (p50/RelA). The heterodimer is sequestered in the cytoplasm in association with inhibitor of NF-xcexaB (IxcexaB)-family of proteins to be kept in an inactive state. IxcexaB-family proteins mask the nuclear translocation signal of NF-xcexaB. Upon stimulation of cells with various cytokines (e.g. TNF-xcex1, IL-1), CD40 ligand, lipopolysaccharide (LPS), oxidants, mitogens (e.g. phorbol ester), viruses or many others. IxcexaB proteins are phosphorylated at specific serine residues, poly-ubiquitinated, and then degraded through a proteasome-dependent pathway. Freed from IxcexaB, the active NF-xcexaB is able to translocate to the nucleus where it binds in a selective manner to preferred gene-specific enhancer sequences. Among the genes being regulated by NF-xcexaB are many coding for pro-inflammatory mediators, cytokines, cell adhesion molecules, and acute phase proteins. Expression of several of these cytokines and mediators in turn can lead to further activation of NF-xcexaB via autocrine and paracrine mechanisms.
Broad evidence is available that suggests a central role of NF-xcexaB in many inflammatory disorders including airway inflammation and asthma ((Yang L et al., J Exp Med 188 (1998), 1739-1750), (Hart L A et al. Am J Respir Crit Care Med 158 (1998), 1585-1592), (Stacey M A et al., Biochem Biophys Res Commun 236 (1997), 522-526) (Barnes P and Adcock I M, Trends Pharmacol Sci 18 (1997), 46-50)).
Further, it has been shown that glucocorticoids, which are by far the most effective treatment for asthma, inhibit airway inflammation by directly interacting with and inhibiting the activity of the transcription factors NF-xcexaB and activating peptide-1 (AP-1) ((Barnes P (1997) Pulmon Pharmacol Therapeut 10, 3-19) and (Dumont A et al. (1998) Trends Biochem Sci 23, 233-235)).
In general, inhibition of NF-xcexaB activation results in strong anti-inflammatory effects similar or superior to those brought upon by steroids. Consequently, NF-xcexaB inhibition should improve inflammatory symptoms typical for asthma; allergic rhinitis; atopic dermatitis; hives; conjunctivitis; vernal catarrh; rheumatoid arthritis; systemic lupus erythematosus; psoriasis; diabrotic colitis; systemic inflammatory response syndrome; sepsis; polymyositis; dermatomyositis; Polyaritis nodoa; mixed connective tissue disease; Sjoegren""s syndrome; gout, and the like.
Further, several studies imply that NF-xcexaB plays an essential role in neoplastic transformation. For example, NF-xcexaB is associated with cell transformation in vitro and in vivo as a result of gene overexpression, amplification, rearrangement, or translocation (Mercurio, F., and Manning, A. M. (1999) Oncogene, 18:6163-6171). In certain human lymphoid tumor cells, the genes of NF-xcexaB family members are rearranged or amplified. Its possible involvement in cancer pathology is also disclosed in Mayo, M. W., Baldwin A. S. (2000) Biochmica et Biophysica Acta 1470 M55-M62. Mayo M. W. et al., discloses the inhibition of NF-xcexaB results in the blockage the initiation and/or progression of certain cancer, particularly colorectal cancer.
Finally, NF-xcexaB may also be involved in the regulation of neuronal cell death. It has been shown that NF-xcexaB becomes activated and promotes cell death in focal cerebral ischemia Nature medicine Vol. 5 No. 5, May 1999).
Extensive research during the past years led to the identification of an IxcexaB kinase (IKK) complex as being responsible for the signal-induced IxcexaB phosphorylation ((Mercurio, F., and Manning, A. M. (1999) Current Opinion in Cell Biology, 11:226-232), (Mercurio, F., and Manning, A. M. (1999) Oncogene, 18:6163-6171), (Barnkett, M., and Gilmore T. D. (1999) Oncogene 18, 6910-6924), (Zandi, E., and Karin, M., (1999) 19:4547-4551), (Israel, A., (2000) trends in CELL BIOLOGY 10:129-133), and (Hatada, E. N, et al. (2000) Current Opinion in Immunology, 12:52-58)). This complex is most likely the site of integration of all of the different stimuli leading to NF-xcexaB activation. The IKK-complex (molecular weight 700-900 kDa) is composed of various proteins including two homologous IxcexaB kinases, called IKK-xcex1 and IKK-xcex2, an upstream kinase, NIK which induces NF-xcexaB, a scaffold protein called IKAP, which tethers together the three kinases, and a regulatory subunit IKK-xcex3, which preferentially interacts with IKK-xcex2.
IKK-xcex2 is a 756 amino acid serine-threonine kinase showing 52% identity to and the same domain structure as IKKxcex1 ((Mercurio F et al (1997) Science 278, 860-866.), (Woronicz J D et al. (1997) Science 278, 866-869.), (Zandi E et al. (1997) Cell 91, 243-252.). IKK-xcex2 forms homo-dimers and hetero-dimers with IKK-xcex1 in vitro and in cells, respectively. IKK-xcex2 also interacts with IKK-xcex3, IKAP, NIK and IxcexaBxcex1. Recombinant IKK-xcex2 phosphorylates IxcexaBxcex1 and IxcexaBxcex2 at specific serine residues with equal efficacy (Li J et al. (1998) J Biol Chem 273, 30736-30741.), (Zandi E, Chen Y, Karin M (1998) Science 281, 1360-1363.). IKK-xcex2 shows a higher constitutive kinase activity as compared to IKK-xcex1. This is in agreement with data suggesting that over-expression of IKK-xcex2 activates the transcription of a NF-xcexaB-dependent reporter gene with a higher efficacy as compared to IKK-xcex1. IKK-xcex2 has been shown to be activated in various cell lines or fresh human cells in response to various stimuli including TNF-xcex1, IL-xcex2, LPS, anti-CD3/anti-CD28 co-stimulation, protein kinase C and calcineurin, B-cell receptor/CD40 ligand stimulation, and vanadate. IKK-xcex2 is activated in fibroblast-like synoviocytes (FLS) isolated from the synovium of patients suffering from rheumatoid arthritis or osteoarthritis (Zandi E et al. (1997) Cell 91, 243-252.), (O""Connell M A et al. (1998) J Biol Chem 273, 30410-30414.), Kempiak S J et al. (1999) J Immunol 162, 3176-3187.). Furthermore, IKK-xcex2 can be activated by the structurally related upstream kinases MEKK-1 and NIK, most likely through phosphorylation of specific serine residues within the T-loop (activation loop) and by certain protein kinase C isoforms ((Nakano H et al. (1998) Proc Natl Acad Sci USA 95, 3537-3542.), (Lee F S et al. (1998) Proc Natl Acad Sci USA 95, 9319-9324.), (Nemoto S et al (1998) Mol Cell Biol 18, 7336-7343.), (Lallena M J et al. (1999) Mol Cell Biol 19, 2180-2188.)). A catalytically inactive mutant of IKK-xcex2 has been shown to inhibit activation of NF-xcexaB by TNF-xcex1, IL-1xcex2, LPS, anti-CD3/anti-CD28 stimulation ((Mercurio F et al. (1997) Science 278, 860-866.), (Woronicz J D et al. (1997) Science 278, 866-869.)). The same effects are observed when MEKK1 or NIK are overexpressed. Additionally, IKK-xcex2 mutations in the activation loop inhibited IL-1 and TNF-xcex1 signaling (Delhase M et al. (1999) Science 284, 309-313.). Based on the experimental results described above, there is clear-cut evidence for a pivotal involvement of IKK-xcex2 in various pathways leading to NF-xcexaB activation.
In summary, the specific inhibition of IKK-xcex2 should result in a strong anti-inflammatory and immuno-modulatory effect in vivo with the potential of improving the underlying causes of asthma and other diseases. In addition, anti-tumor and anti-ischemic effects of an IKK-xcex2 inhibitor may be expected.
Manna et al., disclose 4,6-disubstituted 3-cyano-2-aminopyridines represented by general formulas: 
wherein (Rxe2x80x2, Rxe2x80x3) represent (OCH3, OCH3), (Cl, Cl), (H, Cl), (H, Br), (H, CH3), (H, OCH3), (H, NO2), or (H, N(CH3)2), or 
as a general anti-inflammatory, analgesic, and antipyretic agent (Eur J. Med. Chem. 34, 245-254(1999)).
Manna et al. neither disclose pyridine derivatives with aliphatic groups at position 4 of the pyridine ring, nor suggest IKK-xcex2 kinase or NF-xcexaB inhibitory activity on the above known pyridine derivatives.
The development of a novel compound having effective anti-inflammatory actions based on a specific and selective inhibitory activity to NF-xcexaB has been desired.
As the result of extensive studies on chemical modification of pyridine derivatives, the present inventors have found that the compound of novel chemical structure related to the present invention have unexpectedly excellent IKK-xcex2 kinase inhibitory activity. This invention is to provide the following general formula (I) and the salts thereof: 
wherein xe2x80x94R1 represents 
in which xe2x80x94R11 is hydrogen, C1-6 alkyl halogen, hydroxy, C1-12 alkoxy, nitro, amino, C1-6 alkylsulfonylamino, C1-6 alkoxycarbonyl, C1-6 alkylamino, di(C1-6 alkyl)amino, C1-6 alkanoylamino, phenyl C1-6 alkylamino, phenylsulfonylamino, or xe2x80x94Oxe2x80x94(CH2)nxe2x80x94R111,
wherein n represents an integer selected from 0 to 6, and R111 is C2-6 alkenyl, benzoyl, diphenylmethyl, di(C1-6 alkyl)amino, C1-6 alkanoyl, C1-6 alkoxycarbonyl, or a 3 to 10 membered saturated or unsaturated ring having 0 to 3 heteroatoms selected from the group consisting of S, O and N as heteroatoms and is optionally substituted by C1-6 alkyl, mono or di halogen, halogen substituted C1-6 alkyl, nitro, ciano, C1-6 alkoxycarbonyl, phenyl, hydroxy, amino, C1-6 alkylamino, di(C1-6 alkyl)amino, C1-6 alkanoylamino, C1-6 alkoxy, or carbamoyl;
R2 represents hydrogen or halogen;
R3 represents hydrogen or 1,2,3,6-Tetrahydro-pyridine,
xe2x80x94CR31R32R33,
xe2x80x83wherein R31 is hydrogen or C1-6 alkyl,
R32 is hydrogen, xcex1-aminobenzyl, C1-6 alkyl optionally substituted by one or two substituents selected from the group consisting of hydroxy, amino, amino substituted phenyl, phenyl, halogen substituted phenyl, and C1-6 alkoxy substituted phenyl, or a 5 to 8 membered saturated ring having 0 to 3 atoms selected from the group consisting of S, O and N as heteroatoms and optionally substituted by C1-6 alkyl, and
R33 is hydrogen, amino, C1-6 alkoxycarbonylamino, C2-6 alkenyloxycarbonylamino, piperidino-C1-6 alkylcarbonylamino, piperidinyl-C1-6 alkylcarbonylamino, or
R32 and R33 may form, together with the adjacent carbon atom, a 5 to 8 membered saturated ring having 0 to 3 heteroatoms selected from the group consisting of N, O and S as heteroatoms, which ring is optionally substituted by phenyl-C1-6 alkyl, C1-6 alkoxy substituted phenyl-C1-6 alkyl, C1-6 alkyl, amino, ciano, hydroxy, carbamoyl, carboxy, C1-6 alkylamino, C1-6 alkoxycarbonyl, di(C1-6 alkyl)amino, benzylamino, C1-6 alkylsulfonyl, piperidino C1-6 alkyl carbonyl, or optionally fused by benzene; or
xe2x80x94NR34R35,
xe2x80x83wherein R34 is hydrogen or C1-6 alkyl and
R35 is hydrogen, a 5 to 8 membered saturated ring having 0 to 3 heteroatoms selected from the group consisting of N, O and S as heteroatoms, or xe2x80x94(CH2)mxe2x80x94NR351R352 (m represents any of integers from 1 to 6)
wherein R351 represents hydrogen, C1-6 alkyl,
R352 represents hydrogen, C1-6 alkyl, C1-6 alkanoyl, C1-6 alkyl substituted phenyl, benzoyl, C1-6 alkanoyl, phenylaminocarbonyl, phenylsulfonyl, or
R34 and R35 may form, together with the adjacent N atom, a 5 to 8 membered saturated heterocyclic ring, and said ring may optionally contain NH, S or O atom other than the adjacent N atom and optionally substituted by carbamoyl, amino, or C1-6 alkyl;
R4 represents hydroxycarbonyl, C1-6 alkanoyl, carbamoyl, nitro, cyano, carboxyl, C1-6 alkoxycarbonyl, C1-6 alkylcarbamoyl, C1-6 alkylamino, 5 to 10 membered heteroaryl (hydroxy) methyl, 5 to 10 membered heteroaryl-C1-6 alkyl, or methyl substituted by hydroxy and a 5 to 7 membered saturated cyclic ring, C1-6 alkyl optionally substituted by one selected from the group consisting of hydroxy, C1-6 alkoxy, C1-6 alkylsulfonylamino, C1-6 alkylcarbonylamino, C5-10 aryl, C5-10 arylsulfonyl, C5-10 arylsulfanyl, C5-10 aryloxy, imidazolyl, or dioxo substituted pyrolidino-oxy,
xe2x80x94(CH2)pNHCOR41, xe2x80x94CH2)pNHC(xe2x95x90S)R41
xe2x80x83wherein p represents any of integer from 1 to 6 and R41 represents C1-6 alkoxy, amino, phenylamino, C1-6 alkyl, C1-6 alkylamino, di(C1-6 alkyl)amino, C3-10 cycloalkylamino,
R3 and R4 may form, together with the carbon atoms in the pyridine ring, 4 to 10 membered monocycloalkyl or bicycloalkyl optionally interrupted by NH and optionally substituted by benzyl, xe2x95x90NH, or xe2x95x90O;
R5 represents NR51R52,
wherein R51 is hydrogen, C1-6 alkyl,
R52 is hydrogen, C1-6 alkyl, phenyl, benzyl, C1-6 alkanoyl, or NR51 R52 may form saturated 5-6 membered ring optionally contain NH or O as other heteroatom than the adjacent N atom, or
R4 and R5 may form,
xe2x80x94R40xe2x80x94COxe2x80x94NHxe2x80x94,
xe2x80x94R40xe2x80x94SO2xe2x80x94NHxe2x80x94,
xe2x80x94R40xe2x80x94C(xe2x95x90S)xe2x80x94NHxe2x80x94
xe2x80x94R40xe2x80x94CH2xe2x80x94NHxe2x80x94,
xe2x80x83wherein said xe2x80x94R40xe2x80x94 represents xe2x80x94CHR401xe2x80x94Oxe2x80x94, xe2x80x94CH2xe2x80x94NR401xe2x80x94, xe2x80x94COxe2x80x94NR401xe2x80x94, xe2x80x94CH2xe2x80x94CHR401xe2x80x94, xe2x80x94CHxe2x95x90CR401xe2x80x94, (in which R401 is C1-6 alkanoyl, C1-6 alkyl, phenyl, C1-6 alkylsulfonyl, C3-8 cycloalkylaminocarbonyl, hydrogen, halogen, nitro, amino, ciano, benzoylamino, phenylsulfonyl, carbamoyl, hydroxycarbonyl, C1-6 alkoxycarbonyl, C1-12 alkylaminocarbonyl, halogen substituted C1-6 alkylaminocarbonyl, C1-6 alkanoylamino, C1-6 alkylamino, di(C1-6 alkyl)aminocarbonyl, di(C1-6 alkyl)aminoC1-6 alkylaminocarbonyl, hydroindenylaminocarbonyl, diphenylmethylaminocarbonyl, pyrrolidinocarbonyl, C1-6 alkoxy C1-6 alkyl amino carbonyl, morpholinocarbonyl, piperazinocarbonyl, phenylC1-6 alkylaminocarbonyl, hydroxycarbonylC1-6alkyl-aminocarbonyl, C3-8 cycloalkylaminocarbonyl, C3-8 cycloalkylC1-6 alkylamino-carbonyl, hydroxyC1-6 alkylaminocarbonyl, carboxyethylaminocarbonyl, C1-6 alkylsulfonylaminocarbonyl)
xe2x80x94CR41xe2x95x90Nxe2x80x94NHxe2x80x94 (R41 is hydrogen, amino, or C1-6 alkanoylamino),
xe2x80x94CR42xe2x95x90Nxe2x80x94Cxe2x95x90Nxe2x80x94 (R42 is hydrogen or amino).
The compounds of the present invention surprisingly show excellent IKK-xcex2 kinase inhibitory activity and cytokine inhibitory activity. They are, therefore suitable especially as NF-xcexaB inhibitors and in particular for the production of medicament or medical composition, which may be useful to treat NF-xcexaB dependent diseases.
More specifically, since the pyridine derivatives of the present invention inhibit IKK-xcex2 kinase activity, they are useful for treatment and prophylaxis of diseases involving NF-xcexaB activity as follows: inflammatory symptoms including asthma; allergic rhinitis; atopic dermatitis; hives; conjunctivitis; vernal catarrh; chronic arthrorheumatism; systemic lupus erythematosus; psoriasis; diabrotic colitis; systemic inflammatory response syndrome (SIRS); sepsis; polymyositis; dermatomyositis (DM); Polyaritis nodoa (PN); mixed connective tissue disease (MCTD); Sjoegren""s syndrome; gout; and the like.
The compounds of the present invention are also useful for treatment and prophylaxis of diseases like ischemia and tumor, since the diseases also relate to IKK-xcex2 kinase and NF-xcexaB activity.
Preferred compounds of formula (I) are those wherein:
xe2x80x94R1 represents 
xe2x80x83in which R11 is hydrogen, C1-6 alkyl, halogen, hydroxy, C1-12 alkoxy, amino, C1-6 alkanoylamino, phenyl C1-6 alkylamino, phenylsulfonylamino, or xe2x80x94Oxe2x80x94(CH2)nxe2x80x94R111,
wherein n represents an integer selected from 1 to 6, and R111 is C2-6 alkenyl, benzoyl, diphenylmethyl, di(C1-6 alkyl)amino, C1-6 alkanoyl, C1-6 alkoxycarbonyl, or a 3 to 10 membered saturated or unsaturated ring having 0 to 3 heteroatoms selected from the group consisting of S, O and N as heteroatoms and is optionally substituted by C1-6 alkyl, mono or di halogen, halogen substituted C1-6 alkyl, nitro, ciano, C1-6 alkoxycarbonyl, phenyl;
R2 represents hydrogen;
R3 represents hydrogen, 1,2,3,6-tetrahydro-pyridine
xe2x80x94CR31R32R33,
xe2x80x83wherein R31 is hydrogen or C1-6 alkyl,
R32 is hydrogen, xcex1-aminobenzyl, C1-6 alkyl optionally substituted by one or two substituents selected from the group consisting of hydroxy, amino, amino substituted phenyl, phenyl, halogen substituted phenyl, and C1-6 alkoxysubstituted phenyl, or a 5 to 8 membered saturated ring having 0 to 3 atoms selected from the group consisting of S, O and N as heteroatoms and optionally substituted by C1-6 alkyl, and
R33 is hydrogen, amino, C1-6 alkoxycarbonylamino, C2-6 alkenyloxycarbonylamino, piperidino-C1-6 alkylcarbonylamino, or
R32 and R33 may form, together with the adjacent carbon atom, a 5 to 8 membered saturated ring having 0 to 3 heteroatoms selected from the group consisting of N, O and S as heteroatoms, which ring is optionally substituted by phenyl-C1-6 alkyl, C1-6 alkoxy substituted phenyl-C1-6 alkyl, C1-6 alkyl, amino, carboxy, C1-6 alkylamino, C1-6 alkoxycarbonyl, di(C1-6 alkyl)amino, benzylamino, C1-6 alkylsulfonyl, piperidino C1-6alkyl carbonyl, or optionally fused by benzene; or
xe2x80x94NR34R35,
xe2x80x83wherein R34 is hydrogen and
R35 is hydrogen, a 5 to 8 membered saturated ring having 0 to 3 heteroatoms selected from the group consisting of N, O and S as heteroatoms, or xe2x80x94(CH2)mxe2x80x94NR351R352 (m represents any of integers from 1 to 6)
wherein R351 represents hydrogen, C1-6 alkyl,
R352 represents hydrogen, C1-6 alkyl, C1-6 alkanoyl, C1-6 alkylsubstituted phenyl, benzoyl, C1-6 alkanoyl, phenylaminocarbonyl, phenylsulfonyl, or
R34 and R35 may form, together with the adjacent N atom, a 5 to 8 membered saturated heterocyclic ring, and said ring may optionally contain NH, S or O atom other than the adjacent N atom and optionally substituted by carbamoyl, amino, or C1-6 alkyl;
R4 represents hydroxycarbonyl, C1-6 alkanoyl, carbamoyl, cyano, carboxyl, C1-6 alkoxycarbonyl, C1-6 alkylcarbamoyl, C1-6 alkylamino, 5 to 10 membered heteroaryl (hydroxy) methyl, 5 to 10 membered heteroaryl-C1-6 alkyl, or methyl substituted by hydroxy and a 5 to 7 membered saturated cyclic ring, C1-6 alkyl optionally substituted by one selected from the group consisting of hydroxy, C1-6 alkoxy, C1-6 alkylsulfonylamino, C1-6alkylcarbonylamino, C5-10 aryl, C5-10 arylsulfanyl, C5-10 arylsulfenyl, C5-10 aryloxy, imidazolyl, or dioxo substituted pyrolidino-oxy,
xe2x80x94(CH2)pNHCOR41, xe2x80x94(CH2)pNHC(xe2x95x90S)R41
xe2x80x83wherein p represents any of integer from 1 to 6 and R41 represents C1-6 alkoxy, amino, phenylamino, C1-6 alkyl, C1-6 alkylamino, di(C1-6 alkyl)amino, C3-10 cycloalkylamino,
R3 and R4 may form, together with the carbon atoms in the pyridine ring, 4 to 10 membered monocycloalkyl or bicycloalkyl optionally interrupted by NH and optionally substituted by benzyl, xe2x95x90NH, or xe2x95x90O;
R5 represents NR51R52,
wherein R51 is hydrogen, C1-6 alkyl,
R52 is hydrogen, C1-6 alkyl, phenyl, benzyl, C1-6 alkanoyl, or NR51R52 may form piperidino, or
R4 and R5 may form,
xe2x80x94R40xe2x80x94COxe2x80x94NHxe2x80x94, xe2x80x94R40xe2x80x94SO2xe2x80x94NHxe2x80x94,
xe2x80x94R40xe2x80x94C(xe2x95x90S)xe2x80x94NHxe2x80x94 or
xe2x80x94R40xe2x80x94CH2xe2x80x94NHxe2x80x94,
xe2x80x83wherein said xe2x80x94R40xe2x80x94 represents xe2x80x94CHR401xe2x80x94Oxe2x80x94, xe2x80x94CH2-NR401xe2x80x94, xe2x80x94COxe2x80x94NR401xe2x80x94, (in which R401 is hydrogen, C1-6 alkanoyl, C1-6 alkoxycarbonyl, C1-6 alkyl, phenyl, C1-6 alkylsulfonyl, C3-8 cycloalkylaminocarbonyl, C1-6 alkylaminocarbonyl, carbamoyl, di (C1-6 alkyl)aminocarbonyl), xe2x80x94CH2xe2x80x94CHR402xe2x80x94, xe2x80x94CHxe2x95x90CR402xe2x80x94, (in which R402 is hydrogen, halogen, nitro, amino, ciano, benzoylamino, phenylsulfonyl, carbamoyl, hydroxycarbonyl, C1-6 alkoxycarbonyl, C1-12 alkylaminocarbonyl, halogen substituted C1-6 alkylaminocarbonyl, C1-6 alkanoylamino, C1-6 alkylamino, di(C1-6 alkyl)aminocarbonyl, di(C1-6 alkyl)aminoC1-6 alkylaminocarbonyl, hydroindenylaminocarbonyl, diphenylmethylaminocarbonyl, pyrrolidinocarbonyl, C1-6 alkoxy C1-6 alkyl amino carbonyl, morpholinocarbonyl, piperazinocarbonyl, phenylC1-6 alkylaminocarbonyl, C3-8 cycloalkylaminocarbonyl, hydroxycarbonylC1-6 alkylaminocarbonyl, C3-8 cycloalkylC1-6 alkylaminocarbonyl, hydroxyC1-6 alkylaminocarbonyl, carboxyethylaminocarbonyl, methylsulfonylaminocarbonyl,)
xe2x80x94CR41xe2x95x90Nxe2x80x94NHxe2x80x94 (R41 is hydroxy, amino, C1-6 alkanoylamino) or
xe2x80x94CR42xe2x95x90Nxe2x80x94Cxe2x95x90Nxe2x80x94 (R42 is amino)
or a salt thereof.
More preferred compound of formula (I) are those wherein:
xe2x80x94R1 represents 
xe2x80x83in which R11 is hydrogen, C1-12 alkoxy, or xe2x80x94Oxe2x80x94(CH2)nxe2x80x94R111,
wherein n represents an integer selected from 1 to 6, and R111 is phenyl, C3-8 cycloalkyl;
R2 represents hydrogen;
R3 represents 1,2,3,6-tetrahydro-pyridine,
xe2x80x94CR31R32R33,
xe2x80x83wherein R31 is hydrogen, and
R32 and R33 form, together with the adjacent carbon atom, a 5 to 8 membered saturated ring interrupted by NH, which ring is optionally substituted by phenyl-C1-6 alkyl, C1-6 alkoxy substituted phenyl-C1-6 alkyl, C1-6 alkyl, amino, carboxy, C1-6 alkylamino, C1-6 alkoxycarbonyl, di(C1-6 alkyl)amino, benzylamino, C1-6 alkylsulfonyl, piperidino C1-6 alkyl carbonyl, or optionally fused by benzene; or
xe2x80x94NR34R35,
xe2x80x83wherein R34 is hydrogen and
R35 is xe2x80x94(CH2)mxe2x80x94NR351R352 (m represents any of integers from 1 to 6)
wherein R351 represents hydrogen, C1-6 alkyl,
R352 represents hydrogen, C1-6 alkyl, C1-6 alkanoyl, C1-6 alkylsubstituted phenyl, benzoyl, C1-6 alkanoyl, phenylaminocarbonyl, phenylsulfonyl; and
R4 represents cyano, C1-6 alkyl optionally substituted by hydroxy or C1-6 alkoxy, or
xe2x80x94(CH2)pNHCOR41, xe2x80x94(CH2)pNHC(xe2x95x90S)R41
xe2x80x83wherein p represents any of integer from 1 to 6 and R41 represents C1-6 alkoxy, amino, phenylamino, C1-6 alkyl, C1-6 alkylamino, di(C1-6 alkyl)amino, C3-10 cycloalkylamino;
R5 represents amino, or
R4 and R5 may form,
xe2x80x94R40xe2x80x94COxe2x80x94NHxe2x80x94, xe2x80x94R40xe2x80x94SO2xe2x80x94NHxe2x80x94,
xe2x80x94R40xe2x80x94C(xe2x95x90S)xe2x80x94NHxe2x80x94 or
xe2x80x94R40xe2x80x94CH2xe2x80x94NHxe2x80x94,
xe2x80x83wherein said xe2x80x94R40xe2x80x94 represents xe2x80x94CHR401xe2x80x94Oxe2x80x94, xe2x80x94CH2xe2x80x94NR401xe2x80x94, xe2x80x94COxe2x80x94NR401xe2x80x94, (in which R401 is hydrogen, C1-6 alkanoyl, C1-6 alkoxycarbonyl, C1-6 alkyl, phenyl, C1-6 alkylsulfonyl, C3-8 cycloalkylaminocarbonyl, C1-6 alkylaminocarbonyl, carbamoyl, di(C1-6 alkyl)aminocarbonyl), xe2x80x94CH2CHR402xe2x80x94, xe2x80x94CHxe2x95x90CR402xe2x80x94, (in which R402 is hydrogen, halogen, nitro, amino, ciano, benzoylamino, phenylsulfonyl, carbamoyl, hydroxy-carbonyl, C1-6 alkoxycarbonyl, C1-12 alkylaminocarbonyl, halogen substituted C1-6 alkylaminocarbonyl, C1-6 alkanoylamino, C1-6 alkylamino, di(C1-6 alkyl)aminocarbonyl, di(C1-6 alkyl)aminoC1-6 alkylaminocarbonyl, hydroindenylaminocarbonyl, diphenylmethylaminocarbonyl, pyrrolidinocarbonyl, C1-6 alkoxy C1-6 alkyl amino carbonyl, morpholinocarbonyl, piperazinocarbonyl, phenylC1-6 alkylaminocarbonyl, C3-8 cycloalkylaminocarbonyl, hydroxycarbonylC1-6 alkylaminocarbonyl, C3-8 cycloalkylC1-6 alkylaminocarbonyl, hydroxyC1-6 alkylaminocarbonyl, carboxyethylaminocarbonyl, methylsulfonylaminocarbonyl,)
xe2x80x94CR41xe2x95x90Nxe2x80x94NHxe2x80x94 (R41 is hydroxy, amino, C1-6 alkanoylamino) or
xe2x80x94CR42xe2x95x90Nxe2x80x94Cxe2x95x90Nxe2x80x94 (R42 is amino)
or a salt thereof.
The preferable compounds of the present invention are as follows or the salt thereof:
7-(2-hydroxyphenyl)-5-(3-piperidinyl)-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one;
2-amino-6-[2-(benzyloxy)-6-hydroxyphenyl]-4-(3-piperidinyl)nicotinonitrile;
2-amino-6-(2-hydroxy-6-propoxyphenyl)-4-(3-piperidinyl)nicotinonitrile;
2-[6-amino-5-(hydroxymethyl)-4-(3-piperidinyl)-2-pyridinyl]-3-(benzyloxy)phenol;
7-[2-(benzyloxy)-6-hydroxyphenyl]-5-(3-piperidinyl)-1,4-dihydro-2H-pyrido[2,3d][1,3]oxazin-2-one;
2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(3-piperidinyl)nicotinonitrile trifluoroacetate;
7-(2-hydroxyphenyl)-5-(3-piperidinyl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one;
2-amino-6-[2-(cyclobutylmethoxy)-6-hydroxyphenyl]-4-(3-piperidinyl)nicotinonitrile;
2-[6-amino-5-(hydroxymethyl)-4-(3-piperidinyl)-2-pyridinyl]-3-propoxyphenol;
7-(2-hydroxy-6-propoxyphenyl)-5-(3-piperidinyl)-1,4-dihydro-2H-pyrido[2,3d][1,3]-oxazin-2-one;
ethyl 7-(2-hydroxy-6-propoxyphenyl)-2-oxo-5-(3-piperidinyl)-1,2,3,4-tetrahydro-1,8-naphthyridine-3-carboxylate;
7-(2-hydroxy-6-propoxyphenyl)-5-(3-piperidinyl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one;
2-[6-amino-5-(hydroxymethyl)-4-(3-piperidinyl)-2-pyridinyl]-3-(cyclopropylmethoxy)phenol;
2-[6-amino-5-(hydroxymethyl)-4-(4-piperidinyl)-2-pyridinyl]-3-(cyclopropylmethoxy)phenol;
7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-(3-piperidinyl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one;
ethyl 7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-2-oxo-5-(3-piperidinyl)-1,2,3,4-tetrahydro-1,8-naphthyridine-3-carboxylate;
7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-(4-piperidinyl)-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one; 6xe2x80x2-amino-5xe2x80x2-(hydroxymethyl)-4xe2x80x2-(3-piperidinyl)-2,2xe2x80x2-bipyridin-3-ol;
7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-(4-piperidinyl)-3,4-dihydro-1,8-naphthyridin-2(1H)-one;
7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-3-fluoro-5-(3-piperidinyl)-1,8-naphthyridin-2(1H)-one;
7-(2-hydroxy-6-propoxyphenyl)-5-(4-piperidinyl)-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one;
7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-(3-piperidinyl)-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one;
3-(cyclopropylmethoxy)-2-[5-(3-piperidinyl)-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-7-yl]phenol;
2-[6-amino-5-(hydroxymethyl)-4-(3-piperidinyl)-2-pyridinyl]-3-(neopentyloxy)phenol;
2-[6xe2x80x2-amino-5xe2x80x2-(hydroxymethyl)-1,2,5,6-tetrahydro-3,4xe2x80x2-bipyridin-2xe2x80x2-yl]phenol;
7-[2-(cyclopropyhethoxy)-6-hydroxyphenyl]-5-(3-piperidinyl)-1,8-naphthyridin-2(1H)-one;
N-{[2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(3-piperidinyl)-3-pyridinyl]methyl}acetamide;
7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-2-oxo-5-(3-piperidinyl)-1,2-dihydro-1,8-naphthyridine-3-carboxamide;
3-acetyl-7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-(3-piperidinyl)-3,4-dihydropyrido[2,3-d]pyrimidin-2(1H)-one;
2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(4-piperidinyl)nicotinonitrile;
2-amino-4-[(2-aminoethyl)amino]-6-[2-(cyclopropylmethoxy)-6-hydroxy-phenyl]nicotinonitrile;
N-{[2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(3-piperidinyl)-3-pyridinyl]methyl}-Nxe2x80x2-propylurea;
7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-(3-piperidinyl)-3,4-dihydro-pyrido[2,3-d]pyrimidin-2(1H)-one;
ethyl [2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(3-piperidinyl)-3-pyridinyl]methylcarbamate;
2-amino-6-{2-hydroxy-6-[(4-methylpentyl)oxy]phenyl}-4-(4-piperidinyl)nicotinonitrile;
7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-2-oxo-5-(3-piperidinyl)-1,2,3,4-tetrahydro-1,8-naphthyridine-3-carboxamide;
7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-N-isopropyl-2-oxo-5-(3-piperidinyl)-1,2-dihydro-1,8-naphthyridine-3-carboxamide;
ethyl 7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-2-oxo-5-(3-piperidinyl)-1,4-dihydropyrido[2,3-d]pyrimidine-3(2H)-carboxylate;
N-{[2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(3-piperidinyl)-3-pyridinyl]methyl}urea;
2-amino-6-(2-hydroxy-6-propoxyphenyl)-4-(4-piperidinyl)nicotinonitrile;
N-cyclohexyl-7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-2-oxo-5-(3-piperidinyl)-1,4-dihydropyrido[2,3-d]pyrimidine-3(2H)-carboxamide;
2-amino-6-[2-(cyclobutylmethoxy)-6-hydroxyphenyl]-4-(4-piperidinyl)nicotinonitrile;
7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-N,N-dimethyl-2-oxo-5-(3-piperidinyl)-1,4-dihydropyrido[2,3-d]pyrimidine-3(2H)-carboxamide;
2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(1-methyl-3-piperidinyl)nicotinonitrile;
7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-2-oxo-(3-piperidinyl)-1,4-dihydro-pyrido[2,3-d]pyrimidine-3(2H)-carboxamide;
isopropyl [2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(3-piperidinyl)-3-pyridinyl]methylcarbamate;
isopropyl 7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-2-oxo-5-(3-piperidinyl)-1,4-dihydropyrido[2,3-d]pyrimidine-3(2H)-carboxylate;
isobutyl 7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-2-oxo-5-(3-piperidinyl)-1,4-dihydropyrido[2,3-d]pyrimidine-3(2H)-carboxylate;
neopentyl 7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-2-oxo-5-(3-piperidinyl)-1,4-dihydropyrido[2,3-d]pyrimidine-3(2H)-carboxylate; neopentyl [2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-(3-piperidinyl)-3-pyridinyl]methylcarbamate;
2-amino-6-[2-(hexyloxy)-6-hydroxyphenyl]-4-(4-piperidinyl)nicotinonitrile; and
7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-N-ethyl-2-oxo-5-(3-piperidinyl)-1,4-dihydropyrido[2,3-d]pyrimidine-3(2H)-carboxamide
The compound of the formula (I) of the present invention can be, but not limited to be, prepared by combining various known methods. In some embodiments, one or more of the substituents, such as amino group, carboxyl group, and hydroxyl group of the compounds used as starting materials or intermediates are advantageously protected by a protecting group known to those skilled in the art. Examples of the protecting groups are described in xe2x80x9cProtective Groups in Organic Synthesis (2nd Edition)xe2x80x9d by Greene and Wuts.
The compound (I-a) 
wherein X is CH or N, R11, R2, and R3 are the same as defined,
or a salt thereof, can be prepared, for example, by the following reaction A.
The compound of the formula (II) 
in which R11 and R2 are the same as defined above,
are reacted with an aldehyde of the formula R3xe2x80x94CHO(III), a nitrile of the formula NCCH2CN (IV), and an ammonium salt such as ammonium acetate. R3 is the same as defined above. R3xe2x80x2xe2x80x94CHO(IIIxe2x80x2) can be advantageously used instead of R3xe2x80x94CHO(III) in some instances. R3xe2x80x2 can represent esterified R3 by ethyl, tertiary butyl or the like: or other esters or other substituents which can be easily converted to R3 by conventional methods. Hydroxyl group of the compound of the formula (II) is protected by an appropriate protecting group (e.g., benzyl, methoxybenzy, and silyl) during the reaction, and deprotected afterward. R3xe2x80x2 can also be treated by acids to obtain R3. 
In the sketch above, CN at position C-3 can be replaced by carboxylates derived from tertiary alcohol, such as COOtBu with the use of NCCH2xe2x80x94COOtBu (IVxe2x80x2) instead of NCCH2CN (IV). In this case the following compound (I-axe2x80x2) can be obtained. 
The step 1 and step 1xe2x80x2 of the reaction A can be carried out without a solvent or in a solvent including, for instance, ethers, such as dioxane, and tetrahydrofuran; aromatic hydrocarbons such as benzene, toluene and xylene; nitrites such as acetonitrile; amides such as dimethylformamide (DMF) and dimethylacetamide; sulfoxides such as dimethyl sulfoxide, and others.
The reaction temperature can be optionally set depending on the compounds to be reacted. The reaction temperature is usually, but not limited to, about 50xc2x0 C. to 200xc2x0 C. The reaction may be conducted for, usually, 30 minutes to 48 hours and preferably 1 to 24 hours.
The compounds of the general formula (II), (III), (IIIxe2x80x2) can be commercially available, or can be prepared by the use of known techniques.
Step 2 and step 2xe2x80x2 of the reaction A can be carried out for example, under the hydrogen atmosphere with hydrogeneous catalysis, such as Pd-C in a solvent including, for instance, esters, such as ethyl acetate, ethers, such as dioxane, and tetrahydrofuran; aromatic hydrocarbons such as benzene, toluene and xylene; nitrites such as acetonitrile; amides such as dimethylformamide (DMF) and dimethylacetamide; sulfoxides such as dimethyl sulfoxide, and others.
The reaction temperature can be, but not limited to, about 50xc2x0 C. to 200xc2x0 C. The reaction may be conducted for, usually, 30 minutes to 48 hours and preferably 1 to 24 hours.
Step 3 and Step 3xe2x80x2 of the reaction A can be any kind of conventional reaction starting from ester to obtain R3, e.g., acid treatment, alkali treatment, amidation, and hydrogenation: or other reaction such as alkylation or the like to obtain R3.
Alternatively, the compound (I-b) 
wherein X, R2, and R3 are the same as defined above and Y is C1-12 alkyl or R111xe2x80x94(CH2)nxe2x80x94, in which R111 and n are the same as defined above, or a salt thereof can be obtained by the following reaction B.
The compound (I-bxe2x80x2) can be also obtained in the reaction B. 
In reaction B, the compound of the formula (II-a) is reacted with an aldehyde (III), a nitrile (IV) and ammonium acetate under the same condition as the reaction A to obtain the compound of the formula (Vxe2x80x2). The benzyl protecting groups in the compound of the general formula (II-a) can be replaced with any of appropriate protecting group. The protecting group is then removed after the reaction. In the step 4 of the reaction B, the compound (V) is reacted with L-Y, wherein L represents a leaving group, such as halogen atom e.g., chlorine, bromine or iodine atom; C6-C10 arylsulfonyloxy group e.g. benzenesulfonyloxy, polysulfonyloxy, or p-toluene-sulfonyloxy; and C1-C4 alkylsulfonyloxy group, e.g. methanesulfonyloxy and the like. Y represents C1-C6 alkyl, or xe2x80x94(CH2)nxe2x80x94R111 (wherein R111 is the same as defined above). The reaction with the compound (V) and Lxe2x80x94Y can be carried out in a solvent including, for instance, alcohols such as methanol and ethanol; ethers, such as dioxane, and tetrahydrofuran (THF); nitrites such as acetonitrile; amides such as dimethylformamide (DMF) and dimethylacetamide; sulfoxides such as dimethyl sulfoxide, and others. Optionally, two or more of the solvents selected from the listed above can be mixed and used.
The reaction temperature of the reaction between compound (V) and Lxe2x80x94Y can be optionally set depending on the compounds to be reacted. The reaction temperature is usually, but not limited to, about xe2x88x9210xc2x0 C. to 200xc2x0 C. and preferably about 10xc2x0 C. to 80xc2x0 C. The reaction may be carried out for, usually, 30 minutes to 48 hrs and preferably 1 to 24 hrs. The reaction can be advantageously conducted in the presence of a base. Examples of the base include an alkali metal hydride such as sodium hydride or potassium hydride; alkali metal alkoxide such as sodium methoxide or sodium ethoxide; alkali metal hydroxide such as sodium hydroxide or potassium hydroxide; carbonates such as sodium carbonate or potassium carbonate, and hydrogen carbonates such as sodium hydrogen carbonate and potassium hydrogen carbonate; organic amines such as triethylamine.
The step 3 of the reaction B is the same as that of reaction A.
Alternatively, the compound of the formula (I-c) below: 
wherein X, R11, R2, R34 and R35 are the same as defined above, can be advantageously prepared by the following reaction C. 
First, the compound of the formula (VI) may be reacted with carbon disulfide and R60xe2x80x94L (wherein R60 represents C1-6 alkyl and L represents a leaving group as defined above )to obtain the compound of the formula (VII). Benzyl protecting group in the compound of the formula (VI) can be replaced with any of an appropriate protecting group. This reaction may be advantageously conducted in the presence of base, such as the combination of sodium hydride and dimethyl acetamide.
The resulting compound (VII) may be reacted with cyanoacetamide in the presence of a solvent and a base. Then the compound (VIII) may be oxidized to yield the compound (IX). The compound (IX) is then reacted with halogenoacetoamide such as chloroacetamide in the presence of a base in a solvent. The resulting compound (X) is reacted with NHR34R35 (and R35 are the same as defined above). Finally, the generated product is reacted with a base and is deprotected to obtain the compound (I-c).
The solvents used in each process of the reaction include, for instance, ethers, such as dioxane and tetrahydrofuran; aromatic hydrocarbons such as benzene, toluene and xylene; nitrites such as acetonitrile; amides such as dimethylformaide (DMF) and dimethylacetamide; sulfoxides such as dimethyl sulfoxide, and others. The above solvent may be used alone or in combination.
Examples of the base used in the reaction include an alkali metal hydride such as sodium hydride or potassium hydride; alkali metal alkoxide such as sodium methoxide or sodium ethoxide; alkali metal hydroxide such as sodium hydroxide or potassium hydroxide; carbonates such as sodium carbonate or potassium carbonate, and hydrogen carbonates such as sodium hydrogen carbonate and potassium hydrogen carbonate; organic amines such as triethylamine.
The reaction temperature can be optionally set depending on the compound to be reacted. The reaction temperature, unless otherwise stated above, is about 10xc2x0 C. to 200xc2x0 C. Each process of the reaction may be conducted for, usually, 30 minutes to 48 hours and preferably 1 to 24 hours.
Amino group at position 2 of the pyridine ring is, if necessary, modified according to conventional method to prepare other groups such as alkylamino, alkanoylamino, etc.
When R11 is C1-6 alkylsulfonylamino, C1-6 alkylamino, di(C1-6 alkyl)amino, C1-6 alkanoylamino, phenyl C1-6 alkylamino, or phenylsulfonylamino, it is derived from xe2x80x94NH2 with the use of conventional methods during the course of reaction A.
The compounds of the formulas (I-a), (I-b) and (I-c) can be further reacted to modify the substituents at position 2 and position 3 of the pyridine ring to synthesize the desired compounds in the scope of the present invention. Also, in the course of reaction A, B, and C above, the substituents at position 2 and position 3 of the pyridine ring can be modified.
The amino moiety at position 2 can be modified by the conventional methods as follows: 
wherein L is the leaving group and the same as defined above, Z is benzyl, C1-6 alkyl, or phenyl, or NHZ may form saturated 5-6 membered ring optionally contain NH or O as other heteroatom than the adjacent N atom.
In another embodiment, the amino moiety at position 2 can be converted to amide with the use of acid chloride.
The cyano moiety at position 3 can be converted to carbamoyl by the conventional alkaline hydrolysis.
The tertiary butoxy carbonyl at position 3 can be easily modified, by the conventional reactions of ester, to alcohol, carboxyl, and the like. Alcohol or carboxy may be further converted to another substituents by the conventional methods.
In some embodiment, the substituents of the positions 2 and 3 together form ring optionally having substituents. Any conventional method or combination of any conventional methods can be used to form the rings. The examples of forming the rings are shown below. 
Yet, in another embodiment, the substituents of the positions 3 and 4 together form ring optionally having substituents. Any conventional method or combination of any conventional methods can be used to form the rings. The examples of forming the rings are shown below. 
When the compound shown by the formula (I) or a salt thereof has tautomeric isomers and/or stereoisomers (e.g, geometrical isomers and conformational isomers), each of their separated isomer and mixtures are also included in the scope of the present invention.
When the compound shown by the formula (I) or a salt thereof has an asymmetric carbon in the structure, their optically active compounds and racemic mixtures are also included in the scope of the present invention.
Typical salts of the compound shown by the formula (I) include salts prepared by reaction of the compounds of the present invention with a mineral or organic acid, or an organic or inorganic base. Such salts are known as acid addition and base addition salts, respectively.
Acids to form acid addition salts include inorganic acids such as, without limitation, sulfuric acid, phosphoric acid, hydrochloric acid, hydrobromic acid, hydriodic acid and the like, and organic acids, such as, without limitation, p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like.
Base addition salts include those derived from inorganic bases, such as, without limitation, ammonium hydroxide, alkaline metal hydroxide, alkaline earth metal hydroxides, carbonates, bicarbonates, and the like, and organic bases, such as, without limitation, ethanolamine, triethylamine, tris(hydroxymethyl)aminomethane, and the like. Examples of inorganic bases include, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.
The compound of the present invention or a salts thereof, depending on its substituents, may be modified to form lower alkylesters or known other esters; and/or hydrates or other solvates. Those esters, hydrates, and solvates are included in the scope of the present invention.
The compound of the present invention may be administered in oral forms, such as, without limitation normal and enteric coated tablets, capsules, pills, powders, granules, elixirs, tinctures, solution, suspensions, syrups, solid and liquid aerosols and emulsions. They may also be administered in parenteral forms, such as, without limitation, intravenous, intraperitoneal, subcutaneous, intramuscular, and the like forms, well known to those of ordinary skill in the pharmaceutical arts. The compounds of the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using transdermal delivery systems well known to those of ordinary skilled in the art.
The dosage regimen with the use of the compounds of the present invention is selected by one of ordinary skill in the arts, in view of a variety of factors, including, without limitation, age, weight, sex, and medical condition of the recipient, the severity of the condition to be treated, the route of administration, the level of metabolic and excretory function of the recipient, the dosage form employed, the particular compound and salt thereof employed.
The compounds of the present invention are preferably formulated prior to administration together with one or more pharmaceutically acceptable excipients. Excipients are inert substances such as, without limitation carriers, diluents, flavoring agents, sweeteners, lubricants, solubilizers, suspending agents, binders, tablet disintegrating agents and encapsulating material.
Yet, another embodiment of the present invention is pharmaceutical formulation comprising a compound of the invention and one or more pharmaceutically acceptable excipients that are compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Pharmaceutical formulations of the invention are prepared by combining a therapeutically effective amount of the compounds of the invention together with one or more pharmaceutically acceptable excipients therefor. In making the compositions of the present invention, the active ingredient may be mixed with a diluent, or enclosed within a carrier, which may be in the form of a capsule, sachet, paper, or other container. The carrier may serve as a diluent, which may be solid, semi-solid, or liquid material which acts as a vehicle, or can be in the form of tablets, pills powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.
For oral administration, the active ingredient may be combined with an oral, and non-toxic, pharmaceutically-acceptable carrier, such as, without limitation, lactose, starch, sucrose, glucose, sodium carbonate, mannitol, sorbitol, calcium carbonate, calcium phosphate, calcium sulfate, methyl cellulose, and the like; together with, optionally, disintegrating agents, such as, without limitation, maize, starch, methyl cellulose, agar bentonite, xanthan gum, alginic acid, and the like; and optionally, binding agents, for example, without limitation, gelatin, acacia, natural sugars, beta-lactose, corn sweeteners, natural and synthetic gums, acacia, tragacanth, sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like; and, optionally, lubricating agents, for example, without limitation, magnesium stearate, sodium stearate, stearic acid, sodium oleate, sodium benzoate, sodium acetate, sodium chloride, talc, and the like.
In powder forms, the carrier may be a finely divided solid, which is in admixture with the finely divided active ingredient. The active ingredient may be mixed with a carrier having binding properties in suitable proportions and compacted in the shape and size desired to produce tablets. The powders and tablets preferably contain from about 1 to about 99 weight percent of the active ingredient which is the novel composition of the present invention. Suitable solid carriers are magnesium carboxymethyl cellulose, low melting waxes, and cocoa butter.
Sterile liquid formulations include suspensions, emulsions, syrups and elixirs. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable carrier, such as sterile water, sterile organic solvent, or a mixture of both sterile water and sterile organic solvent.
The active ingredient can also be dissolved in a suitable organic solvent, for example, aqueous propylene glycol. Other compositions can be made by dispersing the finely divided active ingredient in aqueous starch or sodium carboxymethyl cellulose solution or in suitable oil.
The formulation may be in unit dosage form, which is a physically discrete unit containing a unit dose, suitable for administration in human or other mammals. A unit dosage form can be a capsule or tablets, or a number of capsules or tablets. A xe2x80x9cunit dosexe2x80x9d is a predetermined quantity of the active compound of the present invention, calculated to produce the desired therapeutic effect, in association with one or more excipients. The quantity of active ingredient in a unit dose may be varied or adjusted from about 0.1 to about 1000 milligrams or more according to the particular treatment involved.
Typical oral dosages of the present invention, when used for the indicated effects, will range from about 0.01 mg/kg/day to about 100 mg/kg/day, preferably from 0.1 mg/kg/day to 30 mg/kg/day, and most preferably from about 0.5 mg/kg/day to about 10 mg/kg/day. In the case of parenteral administration, it has generally proven advantageous to administer quantities of about 0.001 to 100 mg/kg/day, preferably from 0.01 mg/kg/day to 1 mg/kg/day. The compounds of the present invention may be administered in a single daily dose, or the total daily dose may be administered in divided doses, two, three, or more times per day. Where delivery is via transdermal forms, of course, administration is continuous.
The effect of the present compounds was examined by the following assays and pharmacological tests.
(1) Preparation of IKK-xcex2 Kinase Protein.
A cDNA fragment encoding human IKK-xcex2 open reading frame was generated by PCR with the use of a pair of primers designed from the published sequence (Woronicz J D et al. (1997) Science 278, 866-869). A template was obtained from Quickclone cDNA (Clontech) using Elongase(trademark) Amplification kit (Life Technologies). The DNA fragments generated by PCR were gel-purified and subcloned into pBluescript. The cDNA fragment cloned in pBluescript was inserted into pcDNA3.1/His C KpnI/Notl, and transferred into pVL1393 SmaI/XbaI (Pharmingen) to construct a baculovirus transfer vector. Then the vector, together with the linearized baculovirus (BaculoGold(trademark), Pharmingen) was used to transfect Sf21 cells (Invitrogen, San Diego, Calif.). Generated recombinant baculovirus was cloned and amplified in Sf21 cells, grown in TNM-FH insect cell medium (Life Technologies, Inc.) supplemented with 10% FCS, 50 g/ml Gentamycin, 0.1% Pluronic F-68 (Life Technologies, Inc.) as suspension culture (200 ml in 1 L Erlenmeyer flask; 27xc2x0 C.; 130 rpm). Sf21 cells were infected with this amplified virus with a multiplicity of infection of 5 following standard protocols (Crossen R, Gruenwald S (1997) Baculovirus Expression Vector System Instruction Manual, Pharmingen Corporation) and harvested 48 hrs later. The cells were lysed to obtain the produced chimeric protein of IKK-xcex2 kinase fused by histidine (His-tagged IKK-beta).
(2) The Preparation of Purified GST-IxcexaBxcex1 Fusion Proteins
An expression vector containing the nucleotide sequence encoding fusion protein of GST with amino acid residues 1 to 54 of IxcexaBxcex1 under the control of an IPTG-inducible promoter was constructed. The expression vector was introduced in E. coli and the transformant was cultured and lysed to obtain a GST-IxcexaBxcex1 fusion protein. Then the resulting GST-IxcexaBxcex1 fusion protein was purified and biotinated for kinase assay.
(3) The Measurement of IKK-xcex2 Kinase Activity
The 96-well format kinase assay of IKK-xcex2 were performed to test the inhibitory activity of the compounds of the present invention. First, 5 xcexcl of a test compound was put in the presence of 2.5% dimethyl sulfoxide (DMSO) in each well in a U-bottomed 96-well plate (Falcon). For control wells of background (BG) and total phosphorylation (TP), 5 xcexcl of 2.5% DMSO was put. Recombinant IKK-xcex2 (final 0.6 xcexcg/ml) and bio-GST-IxcexaBxcex1 (1-54) (final 0.2 xcexcM) were diluted in 25 xcexcl of 2xc3x97kinase buffer xcex2 (40 mM Tris-HCl, pH 7.6, 40 mM MgCl2, 40 mM xcex2-glycerophosphate, 40 mM p-nitro-phenylphosphate, 2 mM EDTA, 40 mM creatine phosphate, 2 mM DTT, 2 mM Na3VO4, 0.2 mg/ml BSA and 0.8 mM phenylmethylsulfonyl fluoride) and transferred to the 96-well plate. Bio-GST-IxcexaBxcex1 (1-54) in 25 xcexcl of 2xc3x97kinase buffer xcex2 without IKK-xcex2 was transferred to BG wells. Then 20 xcexcl of 12.5 xcexcM ATP, 62.5 xcexcCi/ml [xcex3-33P] ATP (Amersham Pharmacia Biotech) was added and the resulting mixture was incubated for 2 hrs at room temperature. The kinase reactions were terminated by the addition of 150 xcexcl of termination buffer (100 mM EDTA, 1 mg/ml BSA, 0.2 mg NaN3). One handred and fifty xcexcl of the sample were transferred to a streptavidin-coated, white MTP (Steffens Biotechniche Analysen GmbH #08114E14.FWD) to capture the biotinylated substrates. After 1 hr of incubation, non-bound radioactivity was eliminated by washing the wells five times with 300 xcexcl of washing buffer including 0.9% NaCl and 0.1% (w/v) Tween-20 with the use of a MW-96 plate washer (BioTec). The bound radioactivity was determined after the addition of 170 xcexcl MicroScint-PS scintillation cocktail (Packard) using a TopCount scintillation counter.
(1) Preparation of Syk Protein
A cDNA fragment encoding human Syk openreading frame was cloned from total RNA of human Burkitt""s lymphoma B cell lines, Raji (American Type Culture Collection), with the use of RT-PCR method. The cDNA fragment was inserted into pAcG2T (Pharmingen, San Diego, Calif.) to construct a baculovirus transfer vector. Then the vector, together with the linearized baculovirus (BaculoGold(trademark), Pharmingen), was used to transfect Sf21 cells (Invitrogen, San Diego, Calif.).
Generated recombinant baculovirus was cloned and amplified in Sf21 cells. Sf21 cells were infected with this amplified high titer virus to produce a chimeric protein of Syk kinase fused by glutathione-S-transferase (GST).
The resulting GST-Syk was purified with the use of glutathione column (Amersham Pharmacia Biotech AB, Uppsala, Sweden) according to the manufacturer""s instruction. The purity of the protein was confirmed to be more than 90% by SDS-PAGE.
(2) Synthesize of a Peptide
Next, a peptide fragment of 30 residues including two tyrosine residues, KISDFGLSKALRADENYYKAQTHGKWPVKW, was synthesized by a peptide synthesizer. The N-terminal of the fragment was then biotinylated to obtain biotinylated activation loop peptide (AL).
(3) The Measurement of Syk Tyrosine Kinase Activity
All reagents were diluted with the Syk kinase assay buffer (50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 0.1 mM Na3VO4, 0.1% BSA, 1 mM DTT). First, a mixture (35 xcexcl) including 3.2 xcexcg of GST-Syk and 0.5 xcexcg of AL was put in each well in 96-well plates. Then 5 xcexcl of a test compound in the presence of 2.5% dimethyl sulfoxide (DMSO) was added to each well. To this mixture was added 300 xcexcM ATP (10 xcexcl) to initiate the kinase reaction. The final reaction mixture (50 xcexcl) consists of 0.65 nM GST-Syk, 3 xcexcM AL, 30 xcexcM ATP, a test compound, 0.25% DMSO, and a Syk kinase assay buffer.
The mixture was incubated for 1 hr at room temperature (RT), and the reaction was terminated by the addition of 120 xcexcl of termination buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 500 mM NaCl, 0.1% BSA). The mixture was transferred to streptavidin-coated plates and incubated for 30 min. at room temperature to combine biotin-AL to the plates. After washing the plates with Tris-buffered saline (TBS) (50 mM Tris-HCl (H 8.0), 138 mM NaCl, 2.7 mM KCl) containing 0.05% Tween-20 for 3 times, 100 xcexcl of antibody solution consisting of 50 mM Tris-HCl (pH 8.0), 138 mM NaCl, 2.7 mM KCl, 1% BSA, 60 ng/ml anti-phosphotyrosine monoclonal antibody, 4G10 (Upstate Biotechnology), which was labeled with europium by Amersham Pharmacia""s kit in advance, was added and incubated at room temperature for 60 minutes. After washing, 100 xcexcl of enhancement solution (Amersham Pharmacia Biotech) was added and then time-resolved fluorescence was measured by multi-label counter ARVO (Wallac Oy, Finland) at 340 nm for excitation and 615 nm for emission with 400 msec of delay and 400 msec of window.
(1) Preparation of A549 Cells
The A549 human lung epithelium cell line (ATCC #CCL-885) was maintained in Dulbecco""s modified Eagle""s medium (D-MEM, Nikken Biomedical Institute) supplemented with 10% FCS (Gibco), 100 U/ml penicillin, 100 xcexcg/ml streptomycin, and 2 mM glutamine (culture medium). Forty thousand (4xc3x97104) cells (80 xcexcl/well) were seeded in each well of 96 well flat-bottom tissue culture plate (Falcon #3072). The plate was allowed to stand for 2 hrs, thus the cells were adhered to the bottom of each well. To the each well was added 10 xcexcl vehicle (1% DMSO), serial dilutions of test compounds in 1% DMSO, or 5 nM Dexamethasone in 1% DMSO as a reference. The mixture (90 xcexcl/well) was incubated for 1 hr at 37xc2x0 C. After 1 hr, 1 xcexcg/ml TNF-xcex1 (10 xcexcl) in culture medium was added to the mixture to obtain 100 xcexcl of reaction mixture. The reaction mixture was cultured for 24 hrs to stimulate the cells with 100 ng/ml TNF-xcex1. Cells with vehicle without TNF-xcex1 stimulation were also prepared.
(2) Measurement of RANTES Production
Then the concentration of RANTES released from the cells in the supernatants of each well was determined using a quantitative sandwich enzyme immunoassay technique. First, 2 xcexcg/ml mouse anti-huRANTES mAb (RandD Systems, #mAb678) in PBS buffer (pH 7.4, 100 xcexcl) was put in each well of 96-well NUNC fluoro plate (Nalge Nunc, New York USA) (Final 200 ng/well) and the plate was allowed to stand for overnight at 4xc2x0 C. to be coated by the antibody. Each well of the plate was then washed with 350 xcexcl wash buffer (0.05% Tween-20, 0.85% NaCl, and 25 mM Tris/HCl pH 7.4) for three times. Blocking buffer containing 1% BSA (Sigma 99% pure, 100 g), 5% sucrose (Nacalai tesque, 99% pure, 500 g), and 0.02% azide (Nacalai tesque, 100%, 500 g) were added (200 xcexcl) to each well and then the plate was allowed to stand for 4 hours to stabilize the coated antibody. Next, 50 xcexcl supernatants of cell culture prepared in (1) above were put in each well of the 96-well NUNC fluoro plate with coated antibody. Recombinant Human RANTES (Pepro Tech, Inc. #300-06) was used as the standard for the determination of RANTES production (linear range between 1 and 10 ng/ml). Eu-labelled mouse anti-huRANES mAb (60 ng/ml: RandD Systems, #mAb278) in PBS supplemented by 1% BSA and 0.05% Tween 20 was added (50 xcexcl) to each well. The reaction mixtures were incubated at room temperature for 4 hrs. After washing with wash buffer (0.05% Tween-20, 0.85% NaCl, and 25 mM Tris/HCl pH 7.4, 350 xcexcl/well) for 5 times with the use of a Sera Washer (Bio-Tech, #MW-96R), the enhancement solution (DELFIA, #1244-405, 100 xcexcl/well) was added to each well. The plate was incubated for 10 minutes at room temperature with moderate shaking. Fluorescent intensity was measured using a DELFIA fluorimeter (Wallac). Excitation was performed at 340 nm and emission was measured at 615 nm.
(1) Preparation of PBMC
Human PBMC were prepared by first obtaining blood from healthy donors and isolating the cells from the blood. The isolation was done by Ficoll gradient-centrifugation method using Ficoll Pacque (Pharmacia #17-1440-02). Within three hours from donation, the isolated PBMC was used. After three times washing with PBS, PBMC were resuspended with RPMI 1640 (Nikken BioMedical Institute) supplemented with 10% FCS (Gibco), 100 U/ml penicillin, 100 xcexcg/ml streptomycin, and 2 mM glutamine (culture medium). The cells (1xc3x97105 in 150 xcexcl/well) were seeded in each well of 96 well flat-bottom tissue culture plate (Falcon #3072). To the each well was added 20 xcexcl vehicle (1% DMSO), serial dilutions of test compounds in 1% DMSO, or 250 nM Dexamethasone in 1% DMSO as a reference. The mixture (170 xcexcl/well) was incubated for 1 hr at 37xc2x0 C. After 1 hr, 20 ng/ml LPS (30 xcexcl) in culture medium was added to the mixture to obtain 200 xcexcl of reaction mixture. The reaction mixture was cultured for 7 hrs to stimulate the cells with 3 ng/ml LPS. Cells with vehicle without LPS stimulation were also prepared. The supernatants of the reaction mixture were then collected.
(2) Measurement of TNF-xcex1 Production
The TNF-xcex1 concentration in the supernatants was determined using a DuoSet(trademark) ELISA Development Kit (GenzymeTechne, Minneapolis, USA) following the manufacturer""s recommendations. First, 4 xcexcg/ml of mouse anti-human TNF-xcex1 Ab in PBS buffer (100 xcexcl) was put in each well of 96-well plate (NUNC, Maxisorp(trademark)) and the plate was allowed to stand for overnight at 4xc2x0 C. to be coated with the antibody. Each well of the plate was then washed 5 times with 350 xcexcl of wash buffer containing PBS, 0.05% Tween 20 (Nakalai tesque) using Sera Washer (Bio-Tech, #MW-96R). To each well was added 300 xcexcl of 1% BSA (Sigma), 5% sucrose in PBS. After 2 hrs incubation at room temperature, the buffer was discarded, and 50 xcexcl of culture medium was added. Next, 50 xcexcl supernatant of stimulated cell culture prepared (1) above was put in each well of the 96-well plate. Recombinant human TNF-xcex1 (Genzyme Techne) was used as the standard for the determination of TNF-xcex1 production (linear range between 30 and 2,000 xcexcg/ml). The reaction mixtures were incubated for 1 hr at room temperature. After 5 times washing, 100 xcexcl biotinylated goat anti-human TNF-xcex1 antibody (Genzyme Techne, 300 ng/ml) in 0.1% BSA, 0.05% Tween in PBS (Reagent diluent) was added to each well, and incubated at room temperature for 1 hr. After 5 times washing, 100 xcexcl of Streptavidin-conjugated horseradishperoxidase (Genzyme Techne, 1/100 in Reagent diluent) was added to each well. After 20 min, each well of the plate was washed 5 times with wash buffer (350 xcexcl/well). The substrate of hourseradishperoxidase and H2O2 (TMBZ peroxidase detection kit, SUMILON #ML-1120T) were added to the mixture and the mixture was allowed to stand at room temperature. The reaction was terminated after 10 min by adding 2N H2SO4. Optical density at 450 nm was measured with the use of a microplate reader (Labosystems, Multiscan Multisoft). Quantification of TNF-xcex1 production in each sample was performed by comparison of optical densities between each sample and the standard curve.
IL-2 production was measured in Jurkat T cells (E6-1 clone; ATCC # TIB-152) in response to stimulation with anti-CD3/anti-CD28 antibodies.
(1) Preparation of Immobilized Antibodies
First, anti-CD3 antibodies (400 ng/well Nichirei, NU-T3 4 xcexcg/ml in 100 xcexcl Dulbecco""s PBS) were put in each well of 96-well plate (Falcon #3072) and the plate was allowed to stand for 2 hrs at room temperature to be coated with the antibody. Each well of the plate was then washed with 250 xcexcl PBS 3 times.
(2) Preparation of Jurkat Cell Culture
Jurkat T cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin G, and 100 xcexcg/ml streptomycin (culture medium). Two hundred thousand (2xc3x97105) cells (190 xcexcl/well) were seeded in each well of 96-well U-bottom tissue culture plates (Falcon #3077). To each well was added 10 xcexcl vehicle (0.2% DMSO), serial dilution of compounds in 0.2% DMSO, or 25 nM cyclosporin A as a reference in 0.2% DMSO. The mixture (200 xcexcl) was incubated for one hour at 37xc2x0 C. in a humidified 5% CO2 environment.
(3) Stimulation of the Cell
The reaction mixture obtained in (2) (100 xcexcl) was put in the each well of the antibody-immobilized plate prepared in (1). To this well was added anti-CD28 antibodies (Nichirei, KOLT-2, 6 xcexcg/ml in cell culture medium, 50 xcexcl/well) and 2.5 xcexcg/ml goat anti-mouse kappa chain antibodies (Bethyl Laboratories, (Cat #A90-119A) 10 xcexcg/ml in culture medium, 50 xcexcl/well). The reaction mixture in each well was incubated for 24 hrs at 37xc2x0 C. to stimulate cells with immobilized anti-CD3 antibodies (400 ng/well) and anti-CD28 antibodies (1.5 xcexcg/ml), and then to cross-link receptors on the cells with anti-mouse kappa chain antibodies (2.5 xcexcg/ml).
(4) Measurement of IL-2 Production
The supernatants of the reaction mixture were then collected. The IL-2 concentration in the supernatants was determined using a DuoSet(trademark) ELISA Development Kit (GenzymeTechne, Minneapolis, USA) following the manufacturer""s recommendations. First, 2 xcexcg/ml of mouse anti-huIL-2 Ab in PBS buffer (100 xcexcl) was put in each well of 96-well plate (NUNC, Maxisorp(trademark)) and the plate was allowed to stand for overnight at 4xc2x0 C. to be coated with the antibody. Each well of the plate was then washed 5 times with 350 xcexcl of wash buffer containing PBS, 0.05% Tween 20 (Nakalai tesque) using Sera Washer (Bio-Tech, #MW-96R). To each well was added 250 xcexcl of 1% BSA (Sigma) in PBS, 0.05% Tween 20 (dilution buffer). After 2 hrs incubation at room temperature, the buffer was discarded, and 50 xcexcl of culture medium was added. Next, 50 xcexcl supernatant of stimulated cell culture prepared (3) above was put in each well of the 96-well plate with coated mouse anti-huIL-2 antibody. Recombinant Human IL-2 (Genzyme Techne) was used as the standard for the determination of IL-2 production (linear range between 200 and 5,400 xcexcg/ml). The reaction mixtures were incubated for 1 hr at room temperature. After 5 times washing, 100 xcexcl biotinylated rabbit anti-huIL-2 antibody (Genzyme Techne, 1.25 xcexcg/ml) in dilution buffer was added to each well, and incubated at room temperature for 1 hr. After 5 times washing, 100 xcexcl of Streptavidin-conjugated horseradishperoxidase (Genzyme Techne, 1/1000 in dilution buffer) was added to each well. After 20 min, each well of the plate was washed 5 times with wash buffer (350 xcexcl/well). Substrate and H2O2 (TMBZ peroxidase detection kit, SUMILON #ML-1120T) were added to the mixture and the mixture was allowed to stand at room temperature. The reaction was terminated after 10 min by adding 2N H2SO4. Optical density at 450 nm was measured with the use of a microplate reader (Labosystems, Multiscan Multisoft). Quantification of IL-2 production in each sample was performed by comparison of optical densities between each sample and the standard curve.
Eight weeks old BALB/c female mice were placed into two groups, a control group and a treated group. A solution containing 200 xcexcg/mouse of LPS in 0.9% physiological salt was administered by intraperitoneal (ip) injection into the control mice. Mice in the treated group were first injected ip with compounds of the present invention 30 minutes prior to the LPS injection. Under anesthesia with pentobarbital (80 mg/kg, i.p.), blood was collected from the posterior venous cavity of the treated and control mice at 90 min post-LPS injection into 96-well plate containing 2% EDTA solution. The plasma was separated by centrifugation at 1800 rpm for 10 minutes at 4xc2x0 C. and then diluted with four times volumes of phosphate buffer saline (pH 7.4) containing 1% bovine serum albumin. TNF-xcex1 concentration in the sample was determined using an ELISA kit (Pharmingen, San Diego, Calif.)
The mean TNF-xcex1 level in 5 mice from each group was determined and the percent reduction in TNF-xcex1 levels was calculated. The treated mice showed significant decrease in the level of TNF-xcex1 as compared to the control mice. The result indicates that the compounds of the present invention can restrain LPS-induced cytokine activity.
Results in vitro test and Cellular assay result (A549) are shown in Examples and tables of the Examples below. The data corresponds to the compounds as yielded by solid phase synthesis and thus to levels of purity of about 40 to 90%. For practical reasons, the compounds are grouped in four classes of activity as follows:
In vitro IC50=A(=or less than ) 0.5 xcexcM less than B (=or less than ) 2 xcexcM less than C (=or less than ) 10 xcexcM less than D
Cellular IC50=A (=or  less than ) 10 xcexcM less than B (=or  less than ) 10 xcexcM less than C
The compounds of the present invention also show excellent selectivity and strong activity in other cellular activity and in vivo assays.